Antibodies are widely used for diagnosis and treatment. Antibodies may have improved functions or may be provided with new functions by chemical binding with drugs, enzymes, dyes, peptides, aptamers, linkers, toxins, isotopes, etc. In the chemical binding with these substances, functional groups such as OH group of a tyrosine residue, NH3 group of a lysine residue, or a carboxyl group of aspartic acid or glutamic acid of antibodies are involved (literature [Goldenberg D. M. et al., J. Med. (1978) 298:1384-1386; Rainsbury R. M. et al., Lancet (1983) 2:934-938; and Yamada H. et al., Biochemistry (1981) 20:4836-4842]).
However, these functional groups are also present in the antigen-binding sites. Therefore, if the residues in the antigen-binding sites are involved in the chemical binding, affinity of antibody to antigen may be affected. Further, in the binding reaction using cysteine, reduction of disulfide bonds within a chain (polypeptide) of an antibody molecule or between the chains is a prerequisite, which may affect activity of the protein (literature [Stimmel J. B. et al., J. Biol. Chem. (2000) 273: 30445-30450]). Further, proteins may be rendered inactive or non-specific by misfolding or loss of a tertiary structure (literature [Zhang W. et al., Anal. Biochem. (2002) 311:1-9]).
Site-specific conjugation is preferred over random amino modification as it enables chemical modification of a site away from the binding site, promoting complete retention of biological activity and allowing control over the possible number of prosthetic groups added. Cysteine-engineered antibodies have been conjugated through linkers at the newly introduced cysteine thiols with thiol-reactive linker reagents and drug-linker reagents to prepare cysteine-engineered antibody drug conjugates with anti-cancer properties, for example, anti-MUC16 (US 2008/0311134), anti-CD22 (US 2008/0050310), anti-ROBO4 (US 2008/0247951), anti-TENB2 (US 2009/0117100), anti-CD79B (US 2009/0028856; US 2009/0068202) thio ADC. However, one or more antibody heavy-chain and light-chain variable region genes exist in human or other animals, and as a result, each different antibody has a different framework sequence. Accordingly, there is a problem that the cysteine introduction site should be optimized with respect to respective antibodies.